U.S. patent application number 10/139849 was filed with the patent office on 2003-04-24 for polynucleotide encoding a human junctional adhesion protein (jam-2).
This patent application is currently assigned to Texas Biotechnology Corp.. Invention is credited to Barros, Maria Trindad Arrate, Cunningham, Sonia.
Application Number | 20030079238 10/139849 |
Document ID | / |
Family ID | 22534629 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030079238 |
Kind Code |
A1 |
Cunningham, Sonia ; et
al. |
April 24, 2003 |
Polynucleotide encoding a human junctional adhesion protein
(JAM-2)
Abstract
The present invention relates to an isolated and purified
polynucleotide encoding for a human junctional protein.
Inventors: |
Cunningham, Sonia; (Houston,
TX) ; Barros, Maria Trindad Arrate; (Houston,
TX) |
Correspondence
Address: |
WOOD, PHILLIPS, KATZ, CLARK & MORTIMER
500 W. MADISON STREET
SUITE 3800
CHICAGO
IL
60661
US
|
Assignee: |
Texas Biotechnology Corp.
|
Family ID: |
22534629 |
Appl. No.: |
10/139849 |
Filed: |
May 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10139849 |
May 7, 2002 |
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09643929 |
Aug 23, 2000 |
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60150459 |
Aug 24, 1999 |
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Current U.S.
Class: |
800/8 ;
435/252.3; 435/320.1; 435/325; 435/419; 435/456; 435/69.1; 530/350;
530/388.1; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
A01K 2217/05 20130101; C07K 14/70503 20130101; C07K 16/2803
20130101; C07K 2319/30 20130101 |
Class at
Publication: |
800/8 ; 530/350;
536/23.5; 435/69.1; 435/456; 435/325; 435/419; 435/252.3;
435/320.1; 530/388.1 |
International
Class: |
A01K 067/00; C07H
021/04; C07K 014/435; C12P 021/02; C12N 005/06; C07K 016/20; C12N
005/04; C12N 015/86 |
Claims
What is claimed is:
1. An isolated and purified human JAM2 polynucleotide encoding a
human JAM2 polypeptide or fragment thereof
2. An isolated and purified polynucleotide comprising a nucleotide
sequence of SEQ ID NO: 1.
3. An isolated and purified human JAM2 polypeptide or fragment
thereof.
4. An isolated and purified polypeptide comprising an amino acid
sequence of SEQ ID NO: 2.
5. A recombinant vector comprising a human JAM2 polynucleotide or
fragment thereof, said polynucleotide being operatively linked to a
promoter that controls expression of said polynucleotide sequence
and a termination segment.
6. The vector of claim 5 wherein the promoter is a LTR, SV40, E.
coli, lac trp or phage lambda P.sub.L promoter.
7. A host cell comprising the recombinant vector of claim 5.
8. The host cell of claim 7 wherein the host cell is a bacterial
cell, an animal cell or a plant cell.
9. A transgenic mammal comprising the recombinant vector of claim
5.
10. An antibody binding to the polypeptide of claims 3 or 4.
Description
RELATED APPLICATION INFORMATION
[0001] This application claims priority from U.S. patent
application Ser. No. 60/150,459 filed on Aug. 24, 1999.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to molecular biology. More
specifically, the present invention relates to a polynucleotide
which encodes a human junctional adhesion protein, a polypeptide
encoded by said polynucleotide and to recombinant vectors
expressing said polypeptide.
BACKGROUND OF THE INVENTION
[0003] Cell adhesion is of prime importance for the formation and
functional maintenance of multicellular organisms. Adhesion
proteins can be classified as cell surface molecules that mediate
intercellular bonds and/or participate in cell-substratum
interactions. Their intracellular domains provide a functional link
to the cytoskeleton and this appears to be important for efficient
cell-cell adhesion to take place. They are expressed in
characteristic spatiotemporal sequences. Different superfamilies
have been described including immunoglobulin (hereinafter, "Ig"),
cadherin, integrin, selectin (Aplin A E, Howe A, Alahari S K,
Juniano R L (1998) Pharmacol. Rev. 50:197-263). Adhesion proteins
belonging to the immunoglobulin superfamily may operate in both a
homotypic and/or heterotypic manner. The common building block is
the Ig domain and the prototype is neural cell adhesion molecule
(hereinafter, "NCAM") which possesses five Ig domains. This family
participates in diverse biological functions including
leukocyte-endothelial cell interactions, neural crest cell
migration, neurite guidance and tumor invasion.
[0004] It is well demonstrated that during inflammation members of
the Ig superfamily interact with and participate in leukocyte
adhesion, invasion and migration through the vessel wall
(Gonzalez-Amaro R, Diaz-Gonzales F, Sanches-Madrid F, (1998) Drugs
56:977-88). Selectins are involved in the initial interactions
(tethering/rolling) of leukocytes with activated endothelium,
whereas integrins and Ig superfamily CAMs mediate the firm adhesion
of these cells and their subsequent extravasation.
[0005] Tight junctions (hereinafter, "TJ") and adherens junctions
(hereinafter, "AJ") are specialized structures that occur between
opposing endothelial and epithelial cells. They form a
semipermeable intercellular diffusion barrier that is both dynamic
and regulated. Obviously these structures must be disrupted, or
reorganized, in order to facilitate leukocyte passage from the
circulation. The tight junction is the most apical component of the
junctional complex. In recent years, two types of transmembrane
protein, namely occluding and claudins, have been described that
constitute the tight junction (Fanning A S, Mitic L L, Anderson J
M, (1999) J. Am. Nephrol. 10:1337-45). The possess four putative
transmembrane domains and occluding itself can function as an
adhesion molecule. Occludin directly interacts with ZO-1, a member
of the membrane-associated guanylate kinases (Furuse M, Itoh M,
Hirase T, Nagafuchi A, Yonemura S, Tsukita S, (1994) J. Cell. Biol.
127:1617-26). ZO-1 provides a connection to the perijunctional
cytoskeleton through its ability to associate with actin filaments
(Itoh M, Nagafuchi A, Moroi S, Tsukita S, (1997) J. Cell. Biol.
138(1):181-92)
[0006] The platelet endothelial cell adhesion molecule, PECAM-1, a
member of the Ig superfamily of adhesion proteins, localizes to the
lateral membranes between endothelial cells (Zocchi M R, Ferrero E,
Leone B E, Rovere P, Bianchi E, Toninelli E, Pardi R, (1996) Euro.
J. Immunol. 26:759-67). However, it is not associated with the TJ
and AJ structures (Ayalon O, Sabanai H, Lampugnani M G, Dejana E,
Geiger B (1994) J. Cell Biol. 126(1):247-58). The crucial role of
PECAM-1 in paracellular migration of leukocytes to extravascular
sites has been established (Muller W A, Weigl S A, Deng X, Phillips
D M, (1993), J. Exp. Med. 178:449-60). In 1998 a novel mouse
junctional adhesion molecule (hereinafter, "JAM") was cloned and
identified as an additional transmembrane protein component of the
tight junction (Martin-Padura I, Lostaglio S, Schneemann M,
Williams L, Romano M. Fruscella P, Panzeri C, Stoppacciaro A, Ruco
L, Villa A, Simmons D, Dejana E, (1998) J. Cell. Biol.
(142(1):117-27). JAM possesses two Ig domains; a single
transmembrane and a short intra cellular domain. Thus it belongs to
the Ig superfamily of adhesion molecules and evidence suggests that
it influences the paracellular transmigration of immune cells.
Whether its extracellular domain engages in heterotypic
interactions remains to be elucidated. Nevertheless, the ability to
inhibit JAM function may allow alleviation of inflammatory diseases
such as arthritis, asthma, rheumatoid arthritis, HBD and
Crohns.
[0007] Tight junctions are crucial structures for maintenance of
the blood-brain (hereinafter, "BBB") and blood-retinal
(hereinafter, "BRB") barriers. In some instances it may be
desirable to selectively disrupt endothelial TJs. For example
disruption of the BBB may provide a method for transvascular
delivery of therapeutic agents to the brain. (Muldoon L L, Pagel M
A, Kroll R A, Roman-Goldstein S, Jones R S, Neuwelt E A, (1999) Am.
J. Neuroradiol. 20:217:22). In another instance, strategies
designed to open the tight junctions of polarized epithelial cells
may improve gene delivery for diseases such as cystic fibrosis:
here the polarized apical membranes of airway epithelial cells are
resistant to transfection by lipid:pDNA complexes (Chu Q,
Tousignant J D, Fang S, Jiang C, Chen L H, Cheng S H, Scheule R K,
Eastman S J, (1999) Hum. Gene. Ther. 10:25-36).
SUMMARY OF THE INVENTION
[0008] The present invention relates to an isolated and purified
human JAM2 polynucleotide encoding a human JAM2 polypeptide or
fragment thereof. Moreover, the present invention further relates
to an isolated and purified polynucleotide having the nucleotide
sequence of SEQ ID NO: 1.
[0009] The present invention also relates to an isolated and
purified human JAM2 polypeptide or fragment thereof. Moreover, the
present invention relates to an isolated and purified polypeptide
having the amino acid sequence of SEQ ID NO: 2.
[0010] The present invention also relates to a recombinant vector.
This vector contains a polynucleotide having the nucleotide
sequence of SEQ ID NO: 1, which encodes for a human junctional
adhesion protein. The polynucleotide is operatively linked to a
promoter that controls expression of the nucleotide sequence and a
termination segment.
[0011] The present invention also relates to a host cell containing
recombinant vector. The host cell can be a bacterial cell, an
animal cell or a plant cell. The present invention also relates to
a transgenic mammal containing the recombinant vector described
herein.
[0012] Finally, the present invention relates to an antibody which
binds to the hereinbefore described polypeptide.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1A shows the alignment of a homologous Expressed
Sequence Tag (hereinafter referred to as "EST") obtained from the
databases accessed through the home page of the National Center for
Biotechnology Information at www.ncbi.nlm.nih.gov with the open
reading frame of mouse functional adhesion protein (hereinafter
referred to as "mouse JAM"). FIG. 1B shows the alignment of an
overlapping EST that encodes the 3' end of a human junctional
adhesion protein (hereinafter "human JAM2") including the stop
codon. Identity is shown on the DNA level. FIG. 1C summarizes the
Rapid Amplification of cDNA Ends (hereinafter referred to as
"RACE") procedure employed to obtain the full open reading frame of
human JAM2. The longest clones identified from each reaction are
aligned with mouse JAM.
[0014] FIG. 2 shows the full cDNA and amino acid sequence for the
open reading frame (ORF) of human JAM2. The predicted signal
sequence and transmembrane domain are underlined. N-linked
glycosylation sites are highlighted as are cysteine residues which
form disulfide bonds within the immunoglobulin-like folds in the
extracellular domain. A PKC phosphorylation site is highlighted in
the intracellular domain.
[0015] FIG. 3 shows the alignment of human JAM2 (top) and mouse JAM
(bottom) open reading frames. Conserved cysteine residues predicted
to form disulfide bonds are bolded. Conserved PKC phosphorylation
sited are single underlined.
[0016] FIG. 4 shows identification of the human JAM2 transcript on
normalized multiple tissue Northern blots probed under high
stringency. Transcripts were viewed by hybridization to human JAM2,
actin or GAPDH [.alpha..sup.32P]dCTP labeled probes. FIG. 4 JAM2
(i) and actin (ii) probes: peripheral blood leukocytes (lane 1);
lung (lane 2); placenta (lane 3); small intestine (lane 4); liver
(lane 5); kidney (lane 6); spleen (lane 7); thymus (lane 8); colon
(lane 9); skeletal muscle (lane 10); heart (lane 11); brain (lane
12). FIG. 4B shows JAM2 (i) and GAPDH (ii) probes: right ventricle
(lane 1); left ventricle (lane 2); right atrium (lane 3); left
atrium (lane 4); apex (lane 5); aorta (lane 6); adult heart (lane
7); fetal heart (lane 8). The arrows indicate the human JAM2
transcripts.
[0017] FIG. 5 shows a Western Blot Analysis of JAM2. Cell lysis
from control (lane 1) of JAM2 expressing CHO cells (land 2) was
probed with mouse polyclonal anti-JAM2 extracellular domain
antibody. HSB cell lysis probed with either preimmune (lane 3) or
anti-JAM2 (lane 4) antibody. Equivalent amounts of protein were
loaded in all lanes.
[0018] FIG. 6 shows the localization of JAM2 expressed in Chinese
Hamster Ovary cells by immunofluorescence. Stable cell lines
expressing full-length JAM2 (A) or control (B) were fixed with
paraformaldehyde, stained with 1:100 dilution of primary mouse
anti-JAM2 antibody followed by GAM-FITC. Single angle view of
cellular stained volumetrically reconstructed from 26.times.0.4
.mu.m z-axis planes. Working magnification .times.400. Digital
contrast levels were not changed during image capture. Scale bar,
20 .mu.m.
[0019] FIG. 7 shows screening for JAM2 counter-receptors on various
leukocyte cell lines. Calcein loaded cells were added to JAM2-Fc
captured in 96 well plates. Binding was performed in TBS+Ca/Mg/Mn
(n=6); Wells were washed, retained cells lysed and fluorescence
quantitated with a fluorimeter at excitation 485/emission 530 nm.
Data from a representative experiment. Average.+-.SEM. FU,
arbitrary fluorescence units.
[0020] FIG. 8 shows cation dependence of JAM2 adhesion. Binding of
HSB cells performed in TBS+Ca/Mg/Mn, BB (n=10); TBS (n=7); TBS+EDTA
(N=5); TBS+Ca (n=4); TBS+Mg (n=4); TBS+Mn (n=10). Averaged data
from (n) independent experiments expressed as % Binding Buffer
(BB).+-.SEM. FU, arbitrary fluorescence units. Pairwise
comparisons, by Fisher's PLSD post-hoc test, significantly
different from TBS: *p<0.0001, from TBS+Ca: .sup.tp<0.0001
and from TBS+Mg: .sup..+-.p<0.001.
[0021] FIG. 9 shows the effects of cations on manganese stimulated
JAM2 adhesion. Binding of HSB cells performed in TBS+Ca/Mg/Mn (BB);
TB+Mn; TBS+Mn/Mg; TBS+Mn/Ca. Averaged data from seven (7)
independent experiments expressed as % Binding Buffer (BB).+-.SEM.
FU, arbitrary fluorescence units. Pairwise, comparisons, by
Fisher's PLSK post-hoc test, significantly different from TBS:
*p<0.0001; .paragraph.p<0.01. Significantly, different from
TBS+Mn .sup.Ip<0.001. Significantly different from TBS+Mn/Mg:
.sup.#p<0.01.
[0022] FIG. 10 shows the adhesion of JAM2 Ig domains to HSB cells.
Secreted Fc fusion proteins of JAM2 Ig domain 1, and domains 1+2,
were immobilized on ELISA wells by capture with GAM from the media
of infected SF21 cells.
[0023] FIG. 11 shows the precipitation of surface biotinylated
proteins from HSB cells. Plasma membranes of K562 (lane 1) and HSB
(lanes 2, 3) cells were surface biotinylated and specific binding
proteins precipitated with either JAM2-Fc (lames 1, 2) or JAM1-Fc
(lane 3). JAM1 is the human homologue of mouse JAM, Genbank ACC No.
U89915. Bands were viewed with avidin-HRP and ECL following
electrophoresis and transfer. Equivalent amounts of protein were
loaded in all lanes.
DETAILED DESCRIPTION OF THE INVENTION
[0024] I. The Present Invention
[0025] The present invention relates to an isolated and purified
polynucleotide sequence which encodes for a human junctional
adhesion protein (referred to herein as "human JAM2"). In another
embodiment, the present invention relates to polypeptide for human
JAM2. In yet another embodiment, the present invention relates to
recombinant vectors which, upon expression, produce human JAM2. The
present invention also relates to host cells transformed with these
recombinant vectors.
[0026] II. Sequence Listing
[0027] The present application also contains a sequence listing
that contains 9 sequences. The sequence listing contains nucleotide
sequences and amino acid sequences. For the nucleotide sequences,
the base pairs are represented by the following base codes:
1 Symbol Meaning A A; adenine C C; cytosine G G; guanine T T;
thymine U U; uracil M A or C R A or G W A or T/U S C or G Y C or
T/U K G or T/U V A or C or G; not T/U H A or C or T/U; not G D A or
G or T/U; not C B C or G or T/U; not A N (A or C or G or T/U)
[0028] The amino acids shown in the application are in the L-form
and are represented by the following amino acid-three letter
abbreviations:
2 Abbreviation Amino Acid Name Ala L-Alanine Arg L-Arginine Asn
L-Asparagine Asp L-Aspartic Acid Asx L-Aspartic Acid or Asparagine
Cys L-Cysteine Glu L-Glutamic Acid Gln L-Glutamine Glx L-Glutamine
or Glutamic Acid Gly L-Glycine His L-Histidine Ile L-Isoleucine Leu
L-Leucine Lys L-Lysine Met L-Methionine Phe L-Phenylalanine Pro
L-Proline Ser L-Serine Thr L-Threonine Trp L-Tryptophan Tyr
L-Tryosine Val L-Valine Xaa L-Unknown or other
[0029] III. Polynucleotides
[0030] In one aspect, the present invention provides an isolated
and purified polynucleotide which encodes human JAM2. This
polynucleotide can be a DNA molecule, such as a gene sequence, cDNA
or synthetic DNA. The DNA molecule can be double-stranded or
single-stranded, and if single stranded, may be the coding strand.
In addition, the polynucleotide can be RNA molecules such as
mRNAs.
[0031] The present invention also provides non-coding strands
(antisense) which are complementary to the coding sequences as well
as RNA sequences identical to or complementary to those coding
sequences. One of ordinary skill in the art will readily appreciate
that corresponding RNA sequences contain uracil (U) in place of
thymidine (T).
[0032] In one embodiment, the polynucleotide of the present
invention is an isolated and purified cDNA molecule that contains
the coding sequence of human JAM2. An exemplary cDNA molecule is
shown as SEQ ID NO: 1.
[0033] As is well known in the art, because of the degeneracy of
the genetic code, there are numerous other DNA and RNA molecules
that can code for the same polypeptide as those encoded by SEQ ID
NO: 1 or portions or fragments thereof. The present invention also
contemplates homologous polynucleotides having at least 70%
homology to the sequence shown in SEQ ID NO: 1, preferably at least
80% homology, and most preferably at least 90% homology. The term
"homology", as used herein, refers to a degree of complementarity.
There may be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is one that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid; it is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or Northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous sequence or probe to the target sequence
under conditions of low stringency. This is not to say that
conditions of low stringency are such that non-specific binding is
permitted; low stringency conditions require that the binding of
two sequences to one another be a specific (i.e., selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% identity); in
the absence of non-specific binding, the probes will not hybridize
to the second non-complementary target sequence. Moreover, the
present invention also contemplates naturally occurring allelic
variations and mutations of the cDNA sequences set forth above so
long as those variations and mutations code, on expression, for the
human junctional adhesion protein. The present invention also
encompasses splice variations of the JAM2 polynucleotide.
[0034] The polynucleotide of the present invention can be use din
marker-aided selection using techniques which are well-known in the
art. Marker-aided selection does not require the complete sequence
of the gene. Instead, partial sequences can be used as
hybridization probes or as the basis for oligonucleotide primers to
amplify by PCR or other methods to identify nucleotide specific for
functional adhesion proteins in other mammals.
[0035] IV. Polypeptides
[0036] The present invention also provides for human JAM2
polypeptide. The amino acid sequence for human JAM2 is provided in
SEQ ID NO: 2 and contains 298 amino acid residues.
[0037] The present invention also contemplates amino acid sequences
that are substantially duplicative of the sequences set forth
herein such that those sequences demonstrate like biological
activity to the disclosed sequences. Such contemplated sequences
include those sequences characterized by a minimal change in amino
acid sequence or type (e.g., conservatively substituted sequences)
which insubstantial change does not alter the basic nature and
biological activity of the polypeptide.
[0038] It is well know in the art that modifications and changes
can be made in the structure of a polypeptide without substantially
altering the biological function of the peptide. For example,
certain amino acids can be substituted for other amino acids in a
given polypeptide without any appreciable loss of function. In
making such changes, substitutions of like amino acid residues can
be made on the basis of relative similarity of side-chain
substituents, for example, their size, charge, hydrophobicity,
hydrophilicity, and the like.
[0039] As detailed in U.S. Pat. No. 4,554,101, incorporated herein
by reference, the following hydrophilicity values have been
assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Asp
(+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0);
Pro (-0.5); Thr (-0.4); Ala (-0.5) His (-0.5); Cys (-1.0); Met
(-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5);
and Trp (-3.4). It is understood that an amino acid residue can be
substituted for another having a similar hydrophilicity value
(e.g., within a value of plus or minus 2.0) and still obtained a
biologically equivalent polypeptide.
[0040] In a similar manner, substitutions can be made on the basis
of similarity in hydropathic index. Each amino acid residue has
been assigned a hydropathic index on the basis of its
hydrophobicity and charge characteristics. Those hydropathic index
values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys
(+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8);
Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln
(-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). In
making substitution based on the hydropathic index, a value of
within plus or minus 2.0 is preferred.
[0041] The polypeptide of the present invention can be chemically
synthesized using standard methods known in the art, preferably
solid state methods, such as the methods of Merrifield (J. Am.
Chem. Soc., 85:2149-2154 (1963)). Alternatively, the proteins of
the present invention can be produced using methods of DNA
recombinant technology (Sambrook et al., in "Molecular Cloning--A
Laboratory Manual", 2.sup.nd Ed., Cold Spring Harbor Laboratory
(1989)).
[0042] V. Recombinant Vectors
[0043] The present invention also relates to recombinant vectors
which contain the polynucleotide of the present invention, host
cells which are genetically engineered with recombinant vectors of
the present invention and the production of the polypeptide of the
present invention by recombinant techniques.
[0044] The polynucleotide of the present invention can be employed
for producing polypeptides using recombinant techniques which are
well known in the art. For example, the polynucleotide may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids,
vectors derived from combinations of plasmids and phage DNA, viral
DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
One of the most popular vectors for obtaining genetic elements is
from the well known cloning vector pBR322 (available form the
American Type Culture Collection, Manassas, Va. as ATCC Accession
Number 37017). The pBR322 "backbone" sections can be combined with
an appropriate promoter and the structural sequence can be
expressed. However, any other vector may be used as long as it is
replicable and viable in the, host.
[0045] The polynucleotide sequence of the present invention may be
inserted into one of the hereinbefore mentioned recombinant
vectors, in a forward or reverse orientation. A variety of
procedures, which are well known in the art may be used to achieve
this. In general, the polynucleotide is inserted into an
appropriate restriction endonuclease site(s).
[0046] When inserted into an appropriate expression vector, the
polynucleotide of the present invention is operatively linked to an
appropriate expression control sequence(s), such as a promoter, to
direct mRNA synthesis. As used herein, the term "operatively
linked" includes reference to a functional linkage between a
promoter and a second sequence, wherein the promoter sequence
initiates and mediates transcription of the DNA sequence
corresponding to the second sequence. Generally, operably linked
means that the nucleotide sequences being linked are contiguous
and, where necessary to join two protein coding regions, contiguous
and in the same reading frame. The heterologous structural sequence
can encode a fusion protein including either an N-terminal or
C-terminal identification peptide imparting desired
characteristics, such as stabilization or simplified purification
of expressed recombinant product.
[0047] Promoter regions can be selected from any desired gene using
chloramphenicol transferase (CAT) vectors or other vectors with
selectable markers. Such promoters can be derived from operons
encoding glycolytic enzymes such as 3-phosphoglycerate kinase
(PGK), .alpha.-factor, acid phosphatase, or heat shock proteins.
Examples of bacterial promoters which can be used include, but are
not limited to, lac, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L,
and trp. Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and
mouse metallothionein-I. Examples of other promoters that can be
used include the polyhedron promoter of baculovirus.
[0048] Typically, recombinant expression vectors contain an origin
of replication to ensure maintenance of the vector. They preferably
contain one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells. Examples of
selectable marker genes which can be used include, but are not
limited to, dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, tetracycline or ampicillin resistance for
E. coli. and the TRP1 gene for S. cerevisiae. The expression vector
may also contain a ribosome binding site for translation initiation
and a transcription termination segment. The vector may also
include appropriate sequences for amplifying expression.
[0049] Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described in Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor, N.Y., (1989), which is herein incorporated by reference.
Large numbers of suitable vectors and promoters are commercially
available and can be used in the present invention. Examples of
vectors which can be used include, but are not limited to:
Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript,
psiX174, pBluescript SK, pSKS, pNH8A, pkrH16a, pNH18A, pNH46A
(Stratagene); ptrc99a, PKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); pGEM (Promega). Eukaryotic: pWLNEO, pSV2CAT, p)G44,
pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).
[0050] In another embodiment, the present invention relates to host
cells containing the hereinbefore described recombinant vectors.
The vector (such as a cloning or expression vector) containing the
hereinbefore described polynucleotide, may be employed to
transform, transduce or tranfect an appropriate host to permit the
host to express the protein. Appropriate hosts which can be used in
the present invention, include, but are not limited to prokaryotic
cells such as E. coli., Streptomyces, Bacillus subtilis, Salmonella
typhimurium, as well as various species within the general
Pseudomonas, Streptomyces, and Staphylococcus. Lower eukaryotic
cells such as yeast and insect cells such as Drosophila S3 and
Spodoptera Sf9. Introduction of the recombinant construct into the
host cell can be effected by calcium phosphate transfection,
DEAE-Dextran mediated transfection, or electroporation (see, Davis,
L., Dibner, M., Vattey, L. Basic Methods in Molecular Biology,
(1986), herein incorporated by reference).
[0051] Various higher eukaryotic cells such as mammalian cell
culture systems can also be employed to express recombinant
protein. Examples of mammalian expression systems include the COS-7
lines of monkey kidney fibroblasts, described by Gluzman, Cell,
23:175 (1981), and other cell lines capable of expressing a
compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK
cell lines. Mammalian expression vectors will contain an origin of
replication, a suitable promoter and enhancer, and any necessary
ribosome binding sites, polyadenylation site, splice donor and
acceptor sites, transcriptional termination sequences, and 5'
flanking nontranscribed sequences. DNA sequences derived from the
SV40 splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0052] The engineered host cells can be cultured in conventional
nutrient media modified as appropriate for activating promoters,
selecting transformants or amplifying genes encoding for the human
junctional adhesion protein of the present invention. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression and can
be determined experimentally, using techniques which are well known
in the art.
[0053] Transcription of the polynucleotide encoding the polypeptide
of the present invention by higher eukaryote can be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA which are about from 10 to about 300
base pairs in length, which act on a promoter to increase its
transcription. Examples of suitable enhances which can be used in
the present invention include the SV40 enhancer on the late side of
the replication origin base pairs 100 to 270, a cytomegalovirus
early promoter enhancer, the polyoma enhance on the late side of
the replication origin, and adenovirus enhances.
[0054] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (such as
temperature shift or chemical induction) and cells are cultured for
an additional period. Cells are typically harvested by
centrifugation, disrupted by physical or chemical means, and the
resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted
by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents.
Such methods are well-known to those skilled in the art.
[0055] The polypeptide of the present invention can be recovered
and purified from recombinant cell cultures, the cell mass or
otherwise according to methods of protein chemistry which are known
in the art. For example, ammonium sulfate or ethanol precipitation,
acid extraction, and various forms of chromatography e.g.,
anion/cation exchange, phosphocellulose, hydrophobic interaction,
affinity chromatography including immunoaffinity, lectin and
hydroxylapatite chromatography. Other methods may include dialysis,
ultrafiltration, gelfiltration, SDS-PAGE and isoelectric focusing.
Protein remolding steps can be used, as necessary in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (hereinafter, "HPLC") on normal or reverse
systems or the like, can be employed for final purification
steps.
[0056] Cell-free translation systems can also be employed to
produce such polypeptide using RNAs derived form the DNA constructs
of the present invention.
[0057] The cDNA sequence can be used to prepare stable cell lines
expressing either wt JAM2 or JAM2 mutated at pertinent positions to
determine which part of the molecule is responsible for function.
Stable or transient cell lines can be created with JAM2 processing
a tag at either the 5' or 3' end, e.g., HA epitope, to enable
monitoring of JAM2 function/modification/cellular interactions.
Additionally, cell lines expressing recombinant JAM2, can be used
to screen for small molecule inhibitors of JAM2 function.
[0058] The extracellular sequence of JAM2 can be use to make
recombinant protein fused to the Fe region of mouse/human IgG. This
protein can be used:
[0059] a) To screen for a JAM2 ligand. Briefly, JAM2-Fc fusion can
be captured on ELISA plates. Cultured cells e.g. monocytes can be
labeled with calcine dye, incupated with the immobilized JAM2-Fc,
washed and fluorescence monitored. Alternatively, the JAM2-Fc can
be coupled to a solid support and then used to prepare a column for
purification of solubilized proteins derived from various
cells/tissues. Peptide sequencing could then identify the ligand.
Another approach would be to bind the JAM2-Fc to cell lysates and
perform cross-linking with DSS.
[0060] b) Upon identification of a JAM2 ligand, the JAM2-Fc can be
used to screen for a small molecule inhibitor of JAM2 heterotypic
interactions.
[0061] c) As a tool to neutralize JAM2 function, either heterotypic
or homotypic interactions. The JAM2-Fc may be administered in vivo
in various animal models in order to perturb JAM2 function.
Alternatively, proof of concept studies nay be conducted in
vitro.
[0062] If it is discovered that JAM2 binds in a homotypic manner,
recombinant protein derived from the extracellular domain can be
used to analyze such interactions. Protein would not possess an Fc
Tag. Single immunoglobulin-like domains can be made to determine
which one is responsible for homotypic interactions. Such
recombinant protein can be used to assess its ability to decrease
paracellular permeability in cells expressing native or recombinant
JAM2. The Interactions of the separate domains with each other or
with a recombinant form possessing both Ig-like domains may be
assessed by various means. Examples are cross-linking with DSS,
analytical ultracentrifugation or sizing columns.
[0063] The JAM2 sequence can be used to identify antisense
oligonucleotides for inhibition of JAM2 function in cell systems.
Further, degenerate oligonucleotides may be designed to aid in the
identification of additional members of this family by the
polymerase chain reaction. Alternatively, low stringency
hybridization of cDNA libraries may be performed with JAM2 sequence
to identify closely related sequences.
[0064] The intracellular domain of JAM2 can be used to "fish" for
novel interacting partners in the yeast two-hybrid system. Further,
JAM2 sequence may be use to inactivate an endogenous gene by
homologous recombination and thereby create a JAM2 deficient cell,
tissue or animal. Such cells, tissue or animals may then be used to
define specific in vivo processes normally dependent upon JAM2.
[0065] JAM2 is expressed to a low level in many tissues and it is
likely that JAM2 can be upregulated during pathological conditions.
This expression pattern suggests that JAM2 localizes to
endothelial. However, it is certainly possible that other cell
types also express JAM2. If JAM2 localizes to the tight junction of
epithelial cells, it is proposed that it plays a role during
metastasis. Either defective JAM2 or decreased expression may not
only decrease adhesion between tumor cells but also facilitate
their movement through the endothelium into the vessel. JAM2
expression in the brain may indicate a role in the blood brain
barrier. JAM2 expression in the aorta and heart indicate it may
play a role during conditions which display inflammatory or
permeability changes such as atherogenesis and reperfusion injury.
Further, it is possible that JAM2 localizes to the intercalated
discs of the myocyte and thus play a role in maintenance of the
syncitium.
[0066] VI. Antibodies
[0067] The polypeptide of the present invention, fragments thereof,
or cells expressing said polypeptide can be used as an immunogen to
produce antibodies. These antibodies can be, for example,
polyclonal or monoclonal antibodies. The present invention also
includes chimeric, single chain, and humanized antibodies, as well
as Fab fragments, or the product of a Fab expression library.
[0068] Antibodies generated against the polypeptide of the present
invention can be obtained by administering the polypeptide to an
animal, preferably a nonhuman. Even a sequence encoding only a
fragment of a polypeptide of the present invention can be used to
generate antibodies binding to the whole native polypeptide. Such
antibodies can then be used to isolate the polypeptide from tissue
expressing that polypeptide.
[0069] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (described by
Kohler and Nilstein, 1975, Nature, 256:495-497, herein incorporated
by reference), the tritoma technique, the human B-cell hybridoma
technique (described by Kozbor et al., 1983, Immunology Today,
4:72, herein incorporated by reference), and the EBV-hybridoma
technique to produce human monoclonal antibodies (described by
Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., pp-77-96, herein incorporated by
reference).
[0070] Techniques for the production of single chain antibodies,
such as those described in U.S. Pat. No. 4,946,778, herein
incorporated by reference, can be adapted to produce single chain
antibodies to immunogenic polypeptide of the present invention.
[0071] The antibodies of the present invention can be used to:
[0072] a) Probe cellular localization/expression of JAM2 in tissues
under normal and disease states.
[0073] b) Immunoprecipitate JAM2 protein from cells and/or stroke
tissues to determine whether it is modified by e.g. glycosylation ,
phosphorylation etc.
[0074] c) For helping determine JAM2 function. For example, if it
is found that JAM2 interacts with inflammatory cells or influences
their paracellular migration, neutralizing antibodies will be
developed to inhibit this function both in vitro and in vivo.
[0075] By way of example, and not of limitation, examples of the
present invention shall now be given.
EXAMPLE 1
Cloning and Expression of Human JAM2
[0076] The polynucleotide sequence shown in SEQ IS NO: 1 was cloned
using a combination of electronic and conventional cloning
techniques. The electronic technique used involved utilizing the
Expressed Sequence Tag (EST) databases accessed through the home
page of the National Center for Biotechnology Information (NCBI) at
www.ncbi.nlm.nih.gov. As a template for electronic cloning, the
cDNA sequence of a novel mouse Junctional Adhesion Protein (JAM)
published by Martin-Padura I, Lostaglio S, Schneemann M, Williams
L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa
A, Simmons D, Dejana E, J. Cell. Biol. (1998) 142(1):1 17-27,
herein incorporated by reference, was used. The mouse JAM cDNA
sequence is also available on GenBank (Accession #U89915). The
advance Basic Local Alignment Search Tool (BLAST 2.0) was used to
identify ESTs displaying homology with mouse JAM.
[0077] Electronic Cloning
[0078] The complete mouse JAM peptide sequence (GenBank Accession
#U89915) was searched for homology with human EST sequences using
the tblastn program which compares a protein query sequence against
a nucleotide sequence database dynamically translated in all
reading frames. The complete mouse JAM protein sequence is 300
amino acids in length. The initiation codon begins at base pair
(hereinafter "bp") 71 and the stop codon at 971 (see FIG. 1A). Of
the EST hits, one was chosen for further analysis. The criteria
used were reasonable homology to mouse JAM and conservation of the
systeine residues specific to the immunoglobulin-like fold.
AA406389 and showed 42% identity at the amino acid level with mouse
JAM over a 161 amino acid overlap and was chosen for assembly of a
virtual cDNA (see FIG. 1A). Throughout assembly, translation was
monitored in all reading frames to identify the putative codons for
initiation and termination of the virtual protein. Where possible,
examination of multiple overlapping ESTs was conducted in order to
identify sequencing errors. The final 3' 130 bp of AA406389 was
blasted through the human dbEST using the blastn program. AA912674
showed 99.6% identity over a 257 bp overlap (see FIG. 1B). Further
searching in the database for sequence at the 5'-end of AA406389
did not reveal additional ESTs.
[0079] Conventional Cloning
[0080] In order to obtain further 5' sequence for this cDNA, RACE
was performed. The prime purpose was to identify a putative
ribosome start site (ATG) that coincided approximately in a linear
sequence with that in mouse JAM.
[0081] Three separate RACE reactions were performed consecutively
using the Marathon cDNA amplification kit (Clontech, Palo Alto,
Calif.) on human placental mRNA (Clontech). The first was performed
with an oligonucleotide, 5'-CCCCGCATCACTTCTTGTCACATTTTTGATCCGG-3'
(SEQ ID NO: 3), directed against AA406389. An alignment of this
primer with mouse JAM positioned it some 318 bp downstream of the
transnational start site. mRNA was reverse transcribed with AMV
reverse transcriptase (Clontech) as 42.degree. C. RACE was
performed according to the following protocol:
3 Cycles # Temperature .degree. C. Time 1 94 30 sec 5 94 5 s 72 4
min 5 94 5 s 70 4 min 25 94 5 s 68 4 min
[0082] Products were ligated into the E. coli vector pCRII-TOPO
(Invitrogen, Carlsbad, Calif.) and eleven clones slected for
sequencing (ABI sequencer, Seqwright, Tex.). The longest clone
extended 45 bps 5' of an ATG that approximately aligned with that
of mouse JAM (see FIG. 1C). However, a STOP codon upstream of this
putative translation initiation codon could not be identified.
[0083] In an attempt to identify a STOP codon, in frame and
upstream of this ATG, two additional RACE reactions were performed.
The first used the same primer for extension as RACE reaction 1.
However, mRNA was reverse transcribed with thermoscript (BRL Life
Technologies, New York, U.S.A.) at 58.degree. C. Products were
ligated into pCR-Blunt II--TOPO (Invitrogen). Of six clones
sequenced, the longest only possessed 22 additional base pairs
(FIG. 1C). For RACE reaction 3, an oligonucleotide was designed
within the sequence obtained from RACE reaction 2,
5'-CTGCTCTGAGGAGGTCGAGGGTCCC-3' (SEQ ID NO: 4). The mRNA was
transcribed with thermoscript at 58.degree. C. Three clones were
sequenced and the longest possessed 167 additional base pairs to
that identify in RACE reaction 2 (see FIG. 1C).
[0084] RACE reactions produced in total an additional 234 bp 5' of
the putative translational initiation codon. A stop codon was not
identified within this sequence that was in frame with the ATG.
However, the inventors believe it to be the true start of the open
reading frame for several reasons. First, alignment of the human
JAM2 reading frame with the published mouse JAM reading frame (see
FIG. 3) shows that this ATG approximately coincides with that of
mouse JAM. Second, the nucleotide surrounding this site (GGAAGATGG)
possesses an A at the -3 position and a G at the +4 position thus
conforming to the initiation consensus sequence. Third, the first
28 amino acids of JAM2 predict a single peptide.
[0085] Construction of a Full Length JAM2
[0086] In order to construct full-length human JAM2, the products
of two separate PCT reactions were ligated together via an internal
EcoNI restriction site. For the synthesis of the 5'-section of the
open reading frame, a sense primer encompassing the initiation
codon, 5'-GCCGCGGATCCAAGATGGCGAGGAGG-3' (SEQ ID NO: 5) and an
antisense primer targeted at the end of the extracellular domain,
5'-GCTATTATGCCGGTACCGTTG- AGATCATCTAC-3' (SEQ ID NO: 6), were
designed (restriction sites incorporated into the primers for
subsequent manipulation are underlined). A product was amplified
from human placental mRNA (Clontech) using the following program: 2
min at 95.degree. C., 1 cycle; 20 s at 95.degree. C., 20 s at
58.degree. C., 30 s at 72.degree. C., 35 cycles; 3 min at
72.degree. C., 1 cycle. The approximate 720 bp product was ligated
into pCR II-TOPO.
[0087] For synthesis of the 3'-section of the open reading frame
sense primer, 5'-TAAAAATCGAGCTGAGATGATAG-3' (SEQ ID NO: 7), located
248 bp into the reading frame was coupled with antisense primer,
5'-TTAAATTATAAAGGATTTTGTG-3' (SEQ ID NO: 8), that incorporated the
stop codon (bold). A product was amplified from mRNA derived from
human embryonic kidney cells (HEK-293, available from the American
Type Culture Collection, Manassus, Va., ATCC Accession #CRL-1573)
using the following program: 7 min at 95.degree. C., 1 cycle; 20 s
at 95.degree. C., 20 s at 56.degree. C., 30 s at 72.degree. C., 28
cycles; 5 min at 72.degree. C., 1 cycle. The approximate 649 bp
product was ligated into pCR II-TOPO. Two independent PCR reactions
were performed and a clone for each sequenced for verification of
each base.
[0088] Sequence Features
[0089] The human JAM2 nucleotide and amino acid sequence is shown
in FIG. 2 and in SEQ NOS: 1 and 2, respectively. As shown in FIG.
2, the complete coding region of 298 amino acids features a
putative signal sequence, two immunoglobulin-like domains, a single
transmembrane domain (underlined) and a short intracellular domain.
There are two possible cleavage sites for the signal peptide i.e.
VVA-LG (single underline) or AYG-FS (dotted underline). The
designated ATG is the true transnational initiation signal based on
the fact that it lies with a Kozak consensus and it aligns with
human JAM1 ATG. The four cysteine residues predicted to form
disulfide bonds within the immunoglobulin-like domains are
highlighted. The 1.sup.st and 2.sup.nd cysteine are located in the
first immunoglobulin-like fold and the 3.sup.rd and 4.sup.th in the
second immunoglobulin-like fold. Highlighted are potential N-linked
glycosylation sites (N.times.S/T) at amino acids #98, #187, #236
and a potential PKC phosphorylation site (S/T.times.R/K) at amino
acid #279. Thus JAM2 function may be modified by PKC. Further, the
amino acids at the extreme C-terminus of JAM2 (SFII) conform to a
consensus that would be predicted to interact with PDZ domains
(Songyang Z, Fanning A S, Fu C, Xu J, Marfatia S M, Chishti A H,
Crompton A, Chan A C, Anderson J M, Cantley L C (1997) Science
275:73-77). Proteins containing PDZ domains are predominantly
localized to the plasma membrane and are recruited to specialized
sites of cell-cell contact. Most recently, it has been reported
that the intracellular domain of human JAM (JAM1) binds to the
tight junction associated proteins ZO-1 and AF-6 via their PDZ
domains (Bazzoni G, Martinez-Estrada O M, Orsenigo F, Cordenonsi M,
Citi S, Dejana E, (2000) J. Biol. Chem. 275:20520-20526; Ebnet K,
Schulz C U, Meyer Zu, Brickwedde M K, Pendl G G, Vestweber D.
(2000) J. Biol Chem Jun 15; [epub ahead of print]). Thus it is
highly likely that JAM2 will display similar binding
activities.
[0090] Sequence Alignment
[0091] An alignment of the human junctional adhesion sequence with
mouse JAM reveals 43% similarity and 35% identity at the amino acid
level (see FIG. 3). The positions of the conserved cysteine are
highlighted in both sequences.
[0092] Expression Pattern
[0093] Tissue expression of JAM2 was examined on a normalized human
Multiple Tissue Northern blot (Clontech) with an
[.alpha..sup.32P]dCTP labeled probe derived from the extracellular
domain. The results show that JAM2 is expressed as two transcripts
of approximately 4.5 kb and 1.5 kb (see FIG. 4). The blots were
probed at high stringency and thus these two species likely
represent alternatively spliced products. FIG. 4 shows that human
JAM2 is abundantly expressed in the heart. Expression also occurs
in the placenta with much lower levels apparently in brain and
skeletal muscle. FIG. 4B shows a more detained examination of the
JAM2 transcript in the heart. A clear chamber specific expression
was not apparent. Relative to GAPDH, there is somewhat lower
expression in fetal heart. However, major differences in the aorta,
atrium and ventricles were not observed.
4TABLE 1 Expression Characteristics of Human JAM2 mRNA Source EST,
GenBank Acc # RT-PCR Tissue Mix of melanocyte, fetal heart,
AA406389, AA410345 pregnant uterus Mix of fetal liver & spleen
AI052637 Mix of fetal lung, testis, B cell AA912674, AI017553
Embryo (total) W80145 Brain, anaplastic oligodendroma AI199779
Lung, fetal/adult N90730, T89217 Kidney, normal/tumor AA865038,
AA987434 Prostate AI201753 Heart fetal/4 weeks AI40139, AA445150
Mammary (4 weeks) AI54320, AI690843 Testis AA725566 Placenta +
Endothelial Cells Human Umbilical Vein - Human Umbilical Vein,
immortal + (ECV) Human Aortic + Human Cardiac Microvascular +
Epithelial Cells Human embryonic kidney + (HEK-293 Colonic
adenocarcinoma, CaCo-2 v. low
[0094] While not wishing to be bound by any theory, due to the
homology of JAM2 with mouse JAM, the inventors predict that JAM2
localizes to the endothelial cells of these tissues. This is
confirmed by PCR analysis of mRNA derived from human aortic
endothelial cells and cardiac microvascular endothelial cells (see
Table 1, above). Interestingly, a product from human umbilical vein
endothelial cells (hereinafter referred to as "HUVEC") was barely
detectable. Thus, JAM2 expression may be restricted to certain
vascular beds. In addition to the endothelium, mouse JAM is also
expressed in epithelial cells. Using the polymerase chain reaction,
expression in human embryonic kidney cell line (HEK-293) can be
detected but only very low levels in the colonic epithelial cell
line, CaCo2.
[0095] The EST database contains many ESTs that partially encode
the human JAM2 sequence. Table 1 documents the tissues from which
sequence was derived. It does not provide information about the
level of expression in each tissue. The expression pattern is
consistent with that of the mouse JAM, a protein that is specific
to both the endothelium and epithelium.
EXAMPLE 2
Functional Properties of JAM2
[0096] A. Methods
[0097] 1. Expression of Extracellular Domain in Insect Cells
[0098] Oligonucleotides were designed to amplify the extracellular
domain of human JAM2 from the full-length clone. Sense
5'-GCCGCGGATCCAAGATGGCGAG- GAGG-3' (SEQ IS NO: 5) and antisense
5'-GCTATTATGCCGGTACCGTTGAGATCATC-3' (SEQ ID NO: 6) oligonucleotides
incorporated BamHI and KpnI restriction sites (underlined) for
subcloning of the product into a pFastBac1 (Life Technologies,
GIBCO BRL, Grand Island, N.Y.) vector that possessed the constant
region of mouse IgG-2a (Cunningham Sa, Tran T M, Arrate M P, Brock
T A, (1999) J. Biol. Chem. 274:18421-7). This vector drives protein
expression from the polyhedron promoter. The recombinant protein is
secreted from the Sf21 insect cells as a fusion to mIgG2a.
[0099] 2. Expression of the Full Length Clone in Mammalian
Cells
[0100] The full-length clone of JAM2 was modified at its C-terminus
by PCR mutagenesis to incorporate an HA-Tag for detection purposes.
The sense 5'-GCCGCGGATCCAAGATGGCGAGGAGG-3' (SEQ ID NO: 5)
oligonucleotide contained a BamHI site (underlined) for subsequent
manipulation. The antisense
5'-TCAGGCGTAGTCGGGCACGTCGTAGGGTAAATTATAAAGGATTTTGTGTGC-3' (SEQ ID
NO: 9) oligonucleotide incorporated a stop codon (underlined) and
sequence (italics) that specified the HA-tag amino acids,
YPYDVPDYA, (SEQ ID NO: 10) to be inserted. JAM2-HA, modified in the
pGEM-7 (Promega, Madison, Wis.) vector, was digested with BamHI and
XhoI (polylinker) and ligated into the BamHI and XhoI sites of
pcDNA6/V5-His (B) (Invitrogen, Carlsbad, Calif.). This vector
utilizes the CMV promoter to drive protein expression.
[0101] CHO-K1 cells were transected with either 10 .mu.g of vector
possessing no insert, or pcDNA6-JAM2 using FuGENE.TM. 6 reagent
(Roche Diagnostics Corporation, Indianapolis, Ind.). Stable cell
lines, control and JAM2, were selected with 5-10 .mu.g/ml of
Blasticidin. For Western blot analysis, cells were lysed in 1%
TX-100 buffer in the presence of protease inhibitors (cocktail set
III, Calbiochem, La Jolla, Calif.). Some 36 .mu.g of protein was
electrophoresed through 10% polyacrylamide gels and probed with
1:2000.times. dilution of preimmune or anti-JAM2 polyclonal serum.
Specific bands were viewed using enhanced chemiluminescence with
1:30,000.times. dilution of GAM-HRP (Fisher, Pittsburgh, Pa.).
[0102] 3. Chromosomal Localization and Intron/Exon Boundaries
[0103] In order to identify gnomic sequences, the public
non-redundant database was searched using the Blastn program with
JAM2 cDNA sequence. the results required minor manual modification
due to dual designation of isolated bases at the end of some exon
boundaries. The correct designation was based on 5' and 3'
splice-site consensus sequences. It was possible to confirm all
intron/exon boundaries by retrieving identical information from
more than one deposit of gnomic sequence.
[0104] 4. Antibodies
[0105] Female BALB/c mice (8-week-old; Harlan, Indianapolis, Ind.)
were immunized and then boosted 3.times., 28 days apart, by
intraperitoneal and subcutaneous injections of 100 .mu.g purified
JAM2 extracellular domain emulsified with an equal volume of
Freund's adjuvant. Complete Freund's adjuvant was used for the
first immunization and incomplete Freund's adjuvant for subsequent
injections. Serum was collected 10 days following each boost.
[0106] 5. Immunofluorescence
[0107] CHO-K1, control or JAM2 expressing, grown on glass slides to
confluence, were fixed with 1% paraformaldehyde and stained with
1:100.times. dilution of either preimmune or anti-JAM2 mouse
polyclonal serum. GAM-FITC at 1:100.times. was used as secondary.
Fluorescence was viewed using a Noran.TM. Confocal laser-scanning
microscope (Noran Instruments, Middleton, Wis.) equipped with argon
laser and appropriate optics and filter module for FITC detection.
Digital images were obtained at .times.400 using a 0.75N/A Nikon
.times.20 lens. A Z-axis motor attached to the inverted microscope
stage was calibrated to move the plane of focus at 0.4 .mu.m steps
through the sample. Collected 12-bit grey scale images at
512.times.480 resolution, stored on a re-writeable optical hard
disk, were volumetrically reconstructed using the
Image-1/MetamorphTM 3-D module (Universal Imaging Corp., Brandywine
Parkway, Pa.).
[0108] 6. Adhesion Assay
[0109] In vitro adhesion assays were formed in 96 well plates
essentially as described in Todderud, G., J. Leukoc. Biol. 52:85
(1992), herein incorporated by reference. Briefly, 50 .mu.l of goat
anti-mouse IgG2a was coated at 5 .mu.g/ml in PBS and used to
capture 4.8 pmoles of JAM2-Fc or mIgG2a (control). Various
leukocyte cell lines i.e. T lymphocytes, HSB, HPB-ALL; B
lymphocytes, RAMOS; monocytic cells, HL60, THP-1, and the
erythroleukemic, K562 lines were labeled with calcein (Molecular
Probes Inc., Eugene, Ore.) at 50 .mu.g/ml for 25 minutes at
37.degree. C. with 250,000 cells/well in binding buffer that
consisted of Tris buffered saline plus 1 mM each of CaCl.sub.2,
MgCl.sub.2, and MnCl.sub.2. Wells were washed 3.times., lysed with
50 mM Tris (pH 7.5), 5 mM EDTA, 1% NP40, and fluorescence read in a
Cytofluor with excitation at 485/20 nm and emission at 530/25 nm.
Specific binding was calculated as fluorescence with JAM2-Fc minus
fluorescence with mIgG2a. For antibody inhibition, protein captured
on wells or HSB cells were incubated for 30 min at RT in binding
buffer with 1:100.times. dilution of preimmune (normal mouse serum)
or anti-JAM2 mouse polyclonal serum. Following incubation, excess
antibody was removed by washing 3.times. prior to continuation of
the assay. Overall differences among experimental groups for each
parameter were first assessed by one-way analysis of variance
(ANOVA) and individual pair-wise group comparisons were analyzed by
Fisher's protected least significance difference (PLSD) post hoc
test.
[0110] 7. Cell Surface Biotinylation
[0111] HSB or K562 cells were surface biotinylated using EZ-Link
Sulfo-NHS-Biotin (Pierce, Rockford, Ill.) according to the
manufacturer's instructions. Cells (2.5.times.10.sup.7/ml) were
washed 3.times. following incubation with 0.5 mg/ml
Sulfo-NHS-Biotin for 30 min at RT. Cell lysis was achieved in Tris
buffered saline (pH 7.5), 1% Triton X-100, 1 mM MnCl.sub.2, 1 mM
MgCl.sub.2, 1 mM CaCl.sub.2 with the inclusion of Protease
Inhibitor Cocktail Set III (Calbiochem, La Jolla, Calif.). Some 5
.mu.g of JAM-Fc fusion was added to approximately 1 mg of lysis and
incubated at 4.degree. C. ON. Proteins bound to JAM were
precipitated with Protein A sepharose (30 .mu.l), boiled 5 minutes
with 10 mM DTT in SDS sample buffer and separated on 9% SDS gels.
Following transfer to PDVF membrane, biotinylated proteins were
detected using streptavidin-HRP (1:4000) and enhanced
chemiluminescence (ECL) (Amersham Pharmacia Biotech, Piscataway,
N.J.).
[0112] B. Results
[0113] JAM2 was mapped to chromosome 21 at position q21.2 using the
public database. Sequence was retrieved at 100% identity from two
continuous non-overlapping sequences of 100,000 bp each (Accession
No. AP000087.1 and AP000086.1). The coding region of JAM2, which
constitutes 897 bp, is distributed over 10 exons as shown below in
Table 2.
5TABLE 2 Exon Intron 3' splice No. Exon (bp) 5' splice (bp)
nnnnnn/(N) 1 >305 TGGGCT/gtaagt 43,994 ttcag/ATCATA 2 66
ACCAAG/gtacag 5,916 tcctag/AGGCTA 3 108 TTCAAG/gtaagc 3,781
taaaag/GTGATT 4 153 TATTAG/gtgatg 4,767 gttcag/TGGCTC 5 203
ACTCTG/gtaagg 3,290 aaatag/CAATTT 6 100 AAGTAG/gtaagc 3,709
ttccag/ATGATC 7 108 TTTCAA/gtaagt 3,347 ttgtag/AAGAAA 8 16
CTTCCA/gtaagt 2,890 aaacag/GAAGAG 9 43 GAAAAT/gtgagt 2,256
tcctag/GATTTC 10 >221 NNNNNN/(n) *n/N--represents unknown bases
Splice site (/)
[0114] Exon refers to coding exons
[0115] The limits of the JAM2 cDNA sequence shown in FIG. 2 spans
some 74,853 bp of gnomic DNA. Various exons were also found in
AP000223 (coding exon 1), AP000225 (coding exons 2, 3, 4, 5 and 6)
and AP0000226 (coding exons 6, 7, 8, 9 and 10). Since the complete
JAM2 transcript(s) is considerably larger than 897 bp (FIG. 4),
further exons in the untranslated regions remain to be identified
either up and/or downstream. All intron/exon boundaries conform to
the consensus CT/AG rule (see Breathnach, R., et al., Annu. Rev.
Biochem., 50:359(1981)).
[0116] A mouse polyclonal serum was raised against the ectodomain
of JAM2 in order to study protein expression and localization. The
antibody was not useful for studying endogenous levels of JAM2 in
native tissues. To gain further insight, a stable CHO cell line
over-expressing JAM2 was generated. Using these cells detection of
JAM2 protein by Western blot analysis was possible. FIG. 5
estimates the molecular mass of JAM2 to be 48 kDa. This is some 14
kDa larger than the size predicted from the peptide sequence.
Glycosylation of JAM2 on at least one of its three N-lined
glycosylation consensus sites could explain this phenomenon.
[0117] The CHO stable cell line was also used to determine cellular
localization on confluent monolayers. FIG. 6 shows that JAM2
partitions to both surface membranes in addition to sites of
cell-cell contact. The border pattern of staining is identical to
that shown by mouse JAM (JAM1) expressed in CHO cells and
endogenous human JAM (JAM1) in HUVECs (see, Martin-Padura, I., et
al., J. Cell Biol. 142:117 (1998)).
[0118] The capacity of JAM2 extracellular domain to adhere to
various leukocyte cell lines according to a previously established
in vitro binding assay performed under static conditions was next
examined (see Todderud, G., J. Leukoc. Biol. 52:85 (1992)). Calcein
loaded cells were allowed to interact with JAM2-Fc captured in 96
well plates in binding buffer (hereinafter "BB") which contained
TBS plus 1 mM calcium, magnesium and manganese. Non-specific
binding of cells to captured mIgG2a was determined simultaneously
and subtracted. FIG. 7 shows that JAM2-Fc is able to capture the T
lymphocyte cell lines HSB and HPB-ALL quite efficiently compared to
interactions with B lymphocytes (RAMOS) and the monocytic cells
HL60 and THP-1. Binding to the erythroleukemic K562 cell lines was
non-existent.
[0119] To further characterize the adhesion, the cation
independence was investigated. Buffers were modified such that
binding was performed in the presence of no cations or calcium,
magnesium or manganese along (see FIG. 8). There are two components
to the adhesion. Firstly, a cation-independent interaction is
demonstrated by the fact that EDTA does not inhibit binding below
that obtained in the presence of all three cations. Secondly, a
cation dependent interaction is described by a manganese specific
enhancement of binding above that obtained in TBS or TBS+EDTA. This
latter suggests integrin involvement. Since the screen conducted in
FIG. 7 was performed under conditions favorable for
cation-independent binding, all cell interactions in TBS plus
manganese were reanalyzed. Manganese enhanced binding was not
apparent on any of the other cell types.
[0120] The JAM2/HSB manganese stimulated binding component is
virtually abolished in the presence of calcium and magnesium (for
example, in binding buffer). In order to determine if only one or
both of these cations were inhibitory to the manganese
augmentation, assays were performed using various cation
combinations (see FIG. 9). The data show that inclusion of calcium
in the manganese only buffer reduced interactions considerably
(p<0.001). The effect of magnesium was statistically
insignificant.
[0121] Mouse JAM (JAM1) is capable of homotypic interactions. Thus,
it was examined whether JAM2 ectodomain bound HSB cells through
this mechanism. FIG. 4 shows that, unlike human JAM (JAM1), JAM2
does not show expression in peripheral blood leukocytes.
Nevertheless, to verify lack of expression in HSB cells, the mouse
polyclonal serum was used to probe for JAM2 protein expression by
Western blotting. No protein was detected (FIG. 5). As further
proof, the surface JAM2 expression level was compared using the
following more sensitive test. The HSB, control and JAM2 expression
CHO cells were loaded with calcein and incubated with either NMS or
anti-JAM2 serum. Cell surface bound JAM2 antibody was detected by
cell capture in 96 well plates coated with goat anti-mouse
secondary antibodies. Table 3 shows that whilst the anti-JAM2 serum
was effective at capturing CHO cells expressing the JAM2 protein,
no HSB cell binding was apparent.
6TABLE 3 Cell Type Antibody AV .+-. SEM HSB pre 1,877 .+-. 234 JAM2
1,135 .+-. 97 CHO control pre 1,210 .+-. 63 JAM2 1,019 .+-. 44 CHO
JAM2 pre 2,151 .+-. 287 JAM2 112,329 .+-. 4457
[0122] To extend these studies, the ability of the mouse anti-JAM2
serum to neutralize HSB binding to recombinant JAM2 was tested.
Antibody was used to block epitopes on recombinant JAM2 captured on
96 well plates. Table 4 shows that whilst preimmune serum is
ineffective, anti-JAM2 serum successfully prevents HSB binding.
Since relatively high levels of JAM2 are coated on these wells, we
were confident that if low levels were expressed on HSB cells, the
antibody should be capable of producing inhibition when incubated
directly with HSB cells. As predicted, under this experimental
set-up, the anti-JAM2 antibody is unable to inhibit HSB
interactions with recombinant JAM2.
7 TABLE 4 Antibody AV .+-. SEM A) Preincubation with captured
JAM2-Fc Preimmune 1221 .+-. 54 anti-JAM2 5 .+-. 2 B) Preincubation
with HSP cells Preimmune 950 .+-. 45 anti-JAM2 1138 .+-. 33
[0123] Many adhesion proteins belonging to the Ig superfamily
utilizes the most N-terminal Ig domain to achieve adhesion. To
assess the binding capacity of the first Ig domain of JAM2, it was
synthesized as a secreted protein in insect cells and binding
compared with the full extracellular domain. FIG. 10 shows that
this N-terminal Ig-fold of JAM2 is indeed capable of adhering to
HSB cells. Further, the enhancement of binding in the presence of
manganese was also retained.
[0124] The inventors postulate that HSB cells express a
counter-receptor for JAM2. To strengthen this hypothesis, and gain
a preliminary characterization of the protein, the inventors
performed precipitation experiments using JAM2-Fc. HSB cells were
surface biotinylated, washed, lysed and incubated with JAM2-Fc in
binding buffer. Bound proteins were precipitated using protein A
and viewed on Western blots with avidin-HRP. FIG. 11 reveals that
indeed JAM2 can specifically capture a surface protein from HAB
cells of approximately 43 kDa. this band is not apparent in surface
biotinylated K562 cells, in agreement with the cell adhesion
studies described above. Further, human JAM 1-Fc, which is unable
to bind calcein loaded HSB cells, does not precipitate this
protein. The inventors predict that this protein is responsible for
the cation-independent binding of JAM2 to HSB cells.
[0125] The present invention is illustrated by way of the foregoing
description and examples. The foregoing description is intended as
a non-limiting illustration, since many variations will become
apparent to those skilled in the art in view thereof. It is
intended that all such variations within the scope and spirit of
the appended claims be embraced thereby.
[0126] Changes can be made to the composition, operation and
arrangement of the method of the present invention described herein
without departing from the concept and scope of the invention as
defined in the following claims.
Sequence CWU 1
1
* * * * *
References